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In Vitro and in Vivo Antibacterial Activity of Marine Alga Turbinaria Ornata Against Pseu- Domonas Aeruginosa in the Freshwater Prawn Macrobrachium Rosenbergii

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In Vitro and in Vivo Antibacterial Activity of Marine Alga Turbinaria Ornata Against Pseu- Domonas Aeruginosa in the Freshwater Prawn Macrobrachium Rosenbergii Central JSM Biotechnology & Biomedical Engineering Bringing Excellence in Open Access Research Article *Corresponding author Periyakali Saravana Bhavan, Department of Zoology, Bharathiar University, Coimbatore - 641046, Tamil In vitro and In vivo Nadu, India, Tel: 91-422-2428495/91-9842498138; Email: Submitted: 26 September 2017 Antibacterial Activity of Marine Accepted: 13 October 2017 Published: 16 October 2017 Alga Turbinaria ornata against ISSN: 2333-7117 Copyright Pseudomonas aeruginosa © 2017 Bhavan et al. OPEN ACCESS in the Freshwater Prawn Keywords • T. ornata • M. rosenbergii Macrobrachium rosenbergii • P. aeruginosa • Survival 1 1 Gopalan Rajkumar , Periyakali Saravana Bhavan *, Veeran • Brown spot Srinivasan2, Annamalai Asaikutti1, and Rajendran Udayasuriyan1 • Hepatopancreas 1Department of Zoology, Bharathiar University, India • Protein 2Department of Animal Science, Bharathidasan University, India Abstract The in vitro and in vivo antibacterial activity of marine alga, Turbinaria ornata against Pseudomonas aeruginosa on Macrobrachium rosenbergii was studied. The hexanic, acetonic and methanolic extracts of T. ornata were subjected to in vitro study, which showed that the methanolic extract of T. ornata was worked well and produced 24.02 ± 0.47mm zone of inhibition against P. aeruginosa. In the in vivo study, the characteristic appearance of brown/black spots was detected all over the body of M. rosenbergii when the prawns were subjected to immersion in water containing P. aeruginosa (107 cells/mL) and fed with basal diet contained no incorporated T. ornata. Activities of metabolic enzymes (GOT and GPT) and antioxidant enzymes (SOD and catalase), and lipid peroxidation (LPO) were found to be significantly increased in the hepatopancreas of M. rosenbergii due to the infection with P. aeruginosa. These effects were greatly reduced/ neutralized when M. rosenbergii fed with methanolic extract of T. ornata incorporated feed, which in turn ultimately enhanced the survival rate of M. rosenbergii against P. aeruginosa infection. The 2D gel electrophoresis revealed degradation of hepatopancreatic proteins in P. aeruginosa infected M. rosenbergii, whereas the reverse was seen in methanolic extracts of T. ornata incorporated feed fed prawns. Thus, T. ornata possessed the potency of anti P. aeruginosa. One of the active principles of T. ornata, 2-Hexadecen-1-ol, 3,7,11,15-tetramethyl-,[R-[R*,R*-(E)]] was found to be bind with the protein, exotoxin-A of P. aeruginosa when molecular docking was performed. Therefore, there is scope for developing aquaculture medicine with T. ornata against Pseudomonas infection. ABBREVIATIONS The bacterial disease associated with Aeromonas and other PL: Post Larvae; SOD: Superoxide Dismutase; CAT: Catalase; genera of chitinolytic bacteria (Pseudomonas, Vibrio, Beneckea GOT: Glutamic Oxaloacetic Transaminase; GPT: Glutamic Pyruvic and Leucothrix) leaves brown/black spots, necrosis in the Transaminase; LPO: Lipid Peroxidation; 2D gel: 2 Dimensional hepatopancreas and gill disablement [6,7]. A pathogenic Gram Gel Electrophoresis negative bacterium, Pseudomonas aeruginosa can be found in water, soil and sediments [8,9]. Therefore, incidence of P. INTRODUCTION aeruginosa Aquaculture has improved the socio-economic status of prawn M. rosenbergii have been reported [10-13]. Its control is a infections in fish and shellfish including freshwater rural people [1]. The freshwater prawn genus Macrobrachium challenging task. (Palaemonidae), distributed throughout the tropical and About 6000 species of different marine algae have been subtropical zones of the world. Worldwide, 100 species of reported under green, brown and red categories. They are used freshwater prawns have been recorded, among them 40 as food, feed and fertilizer. They have biomedical potentials due species are found in India [2]. Macrobrachium rosenbergii is a potential candidate species for aquaculture, because it provides anti-microbial, anti-viral and anti-tumoral agent [14]. Therefore, nutritious delicacy for human being, and fetched for good price theto presence present ofstudy many aimed bioactive to examine principles the asantibacterial anti-inflammatory, activity with high demand in both domestic and export markets [3]. of the brown alga Turbinaria ornata by their in vitro (by using Disease outbreak due to viral, bacterial and fungal infections hexanic, acetonic and methanolic extracts) and in vivo activity against Pseudomonas aeruginosa induced infection in M. aquaculture, which has become an issue of global concern [4,5]. rosenbergii PL, offering 1.5% methanolic extract of T. ornata threatened the sustainability and profitability of crustacean Cite this article: Rajkumar G, Bhavan PS, Srinivasan V, Asaikutti A, Udayasuriyan R (2017) In vitro and In vivo Antibacterial Activity of Marine Alga Turbi- naria ornata against Pseudomonas aeruginosa in the Freshwater Prawn Macrobrachium rosenbergii. JSM Biotechnol Bioeng 4(2): 1080. Bhavan et al. (2017) Email: Central Bringing Excellence in Open Access incorporated diet. In order to reveal this, morphological changes, Experimental feeds activities of antioxidant enzymes [superoxide dismutase (SOD) The following branded feed basal ingredients (BI) were and catalase (CAT)] and metabolic enzymes [glutamic oxaloacetic used to formulate the experimental feed. For protein sources, transaminase (GOT) and glutamic pyruvic transaminase (GPT)], lipid peroxidation (LPO), 2D-gel pattern of hepatopancreas, (25%) were taken. Wheat bran (10%) was used as carbohydrate and molecular docking of the ligand, 2-Hexadecen-1-ol, fish meal (25%), groundnut oilcake (25%) and soybean meal 3,7,11,15-tetramethyl-,[R-[R*,R*-(E)]] (one of the active principle compound of T. ornata) with the protein, exotoxin-A of P. source. For lipid source, Sunflower oil (2%) was taken. Tapioca aeruginosa were studied. oilcake,flour (5%) soy and bean egg meal,albumin wheat (7%) bran were were used thoroughlyas binding agents.mixed. The powdered basal ingredients, such as fish meal, groundnut MATERIALS AND METHODS Then sterilized water was used to prepare the dough, which was Collection and identification of marine alga oil was added with the dough and mixed well. Then, methanolic extractsteam cooked of T. ornata and cooled was separately at room temperature. incorporated Then with Sunflowerthe dough The marine alga Turbinaria ornata was collected from the intertidal region of Mandapam coast (Lat. 9°17’N; Lon. 79°19’E) egg albumin were added and mixed well. Finally, 1% of vitamin of Gulf of Mannar, south-east coast of Tamil Nadu, India. The B-complexat concentration forte withof 1.5% vitamin and mixedC (BECOSULES well. Then® tapioca flour and Ltd., Navi Mumbai, India) and a pinch of salt were also added and thoroughly mixed. Sterilized water was adequatelyCAPSULES, addedPfizer macroalga species was identified based on its morphology by using published by Central Marine Fisheries Research Institute (ICAR), for maintaining the mixer in moist and paste form. This was Kochi,identification India. Finally, manual the of “Economically species was authenticated Important Seaweeds” by Botanical [15] Survey of India (BSI), Coimbatore, India. The pellets were immediately dried in a thermostatic oven at 37- pelletizedº in a manual pelletizer fixed with 3mm diameter mesh. Preparation of extracts of T. ornata 40 C for one hour to quickly reduce the moisture in order to keep them intact, and then shade dried until they reached constant The collected sample was cleaned well with seawater to weight. To maintain its brittleness and prevent fungal attack they remove all the extraneous matter such as epiphytes, sand particles were kept in air tight jars and stored at -20ºC, and used afresh. The and necrotic parts, and brought to the laboratory in plastic proximate composition of organic matters present in the basal bags. The sample was then thoroughly washed with freshwater, diet formulated was determined by adopting the methodology blotted, spread out and shade dried at room temperature for 2 of Castell and Tiews [18] as given in AOAC [19] manual, which 9% total ash, 8.6 % moisture, 32.9% carbohydrate (total nitrogen weeks,The and powdered was ground sample to fine of powder.T. ornata (75g) was separately freecontains extract) 40.5% and crude gross protein,energy, 4281kcal/kg.5.6% crude fat, 3.4% crude fibre, was done with 450ml (1:6 w/v) of hexane, acetone and methanol Experimental animal packed in Whatmann No. 1 filter paper and Soxhlet extraction individually for 9 h each (36 cycle) until a clear colorless solution The post larvae (PL-10) of the freshwater prawn, Macrobrachium rosenbergii were procured from ADAK Hatchery, muslin cloth, concentrated at 40-50°C using rotary vacuum Odayam, Varkala, Thiruvananthapuram, Kerala, India. They evaporatorwas obtained. (ROTAVAP) These extracts attached were with filtered ultra-cryostat by using double and driedlayer at 40°C under hot air oven [16]. The dark, gummy solid obtained with oxygenated water and acclimatized to ambient laboratory was used for further investigation. conditionswere transported for 2 weeks to inthe cement laboratory tank (6 in × 3polythene × 3 feet) with bags ground filled water (temperature, 27 ± 1.0; pH, 7 ± 0.15; total dissolved solids, Determination of in vitro antibacterial activity 0.9 ± 0.005g L-1; dissolved oxygen, 7.2 ± 0.55mg L-1; BOD 30.0 ± The in-vitro antibacterial activity was studied by agar 1.30mg L-1; COD, 125.0 ± 3.2mg L-1; ammonia, 0.028 ± 0.004mg well diffusion
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